Abstract: Recent studies have exposed a new layer of dynamic complexity in DNA that can potentially redefine our basic view of the structure of DNA in vivo. NMR studies have shown that in canonical duplex DNA, G-C and A-T Watson-Crick base pairs exist in dynamic equilibrium with alternative Hoogsteen base pairs in which the purine flips 180 to form a new set of hydrogen bonds. There are now several documented examples in which Hoogsteen base pairs play crucial roles in DNA based transactions including damage induction, accommodation, and repair, replication, and sequence-specific DNA- protein recognition. Despite their growing importance, visualizing Hoogsteen base pairs in protein-DNA complexes remains an outstanding challenge in structural biology principally as they are very challenging to distinguish from Watson-Crick base pairs using X-ray crystallography. Indeed, we have obtained evidence that many Hoogsteen base pairs in high-resolution X-ray structures of DNA-protein complexes including the nucleosome may have been improperly modeled as Watson-Crick base pairs. This project will develop methods based on (IR) spectroscopy and site-specific modifications to allow the robust structural and functional characterization of Hoogsteen base pairs in large DNA-protein complexes. These methods will be used to test the hypothesis that the nucleosome particle is enriched with Hoogsteen base pairs that contribute to sequence-specific nucleosome stability. Aim 1 will use NMR and X-ray crystallography to determine IR signatures for Hoogsteen base pairs in model duplexes. Aim 2 will use IR and isotope-labeling to resolve suspected Hoogsteen base pairs in X-ray structures of the nucleosome core particle. Aim 3 will examine a broader range of nucleosome positioning sequences to determine how the Hoogsteen levels vary with sequence and will also assess the functional importance of these Hoogsteen base pairs by examining the impact of site- specific 7-deazopurine and ribonucleotide substitutions on nucleosome stability. !